CN102749456B - Kit for chemilumineseent quantitative immunoassay of angiotensin I and preparation method thereof - Google Patents
Kit for chemilumineseent quantitative immunoassay of angiotensin I and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kit for chemilumineseent quantitative immunoassay of an angiotensin I. The kit comprises an Ang I antibody-coated plate, a horse radish peroxidase-labeled Ang I, an Ang I calibrator, Ang I quality control materials, a light-emitting solution A, a light-emitting solution B, and a washing liquid concentrated by 20 times. The invention also discloses a preparation method of the kit for chemilumineseent quantitative immunoassay of an angiotensin I. Compared with the existing kit, the kit provided by the invention can be operated more simply, is safe and does not produce pollution on the environment. In addition, the kit provided by the invention allows a wide range of tested sample concentrations and has a long reagent validity period and good stability.
Description
Technical field
The present invention relates to immunoassay medical domain, be specifically related to angiotensinⅠ (Ang I) chemiluminescence immunoassay immue quantitative detection reagent box and preparation method thereof.
Background technology
Hypertension (Hypertension) is a kind of global common disease, and morbidity rate is all over the world up to 10% ~ 20%, and can cause the pathology of the cerebrovascular, heart, kidney, is the principal disease of harm humans health.According to Ministry of Public Health's latest data, the existing hyperpietic of China more than 200,000,000, along with economic development, the raising of living standards of the people, hypertension becomes an important public health problem day by day.
AngiotensinⅠ (Angiotensin I, Ang I) is by former formation of feritin vasoactive Angiotensin Converting Enzyme.Peptide bond hydrolysis between feritin energy catalysis angiotensins norleucine (Leu) and valine (Val) produces decapeptide angiotensinⅠ.AngiotensinⅠ does not have biologic activity under normal plasma concentration, but exists as the precursor of angiotensinⅡ.AngiotensinⅠ can stimulate adrenal medella secretion adrenaline, and its direct pressor effect is not obvious; AngiotensinⅡ can make whole body parteriole shrink and rising blood pressure, in addition, also can promote adrenal cortex secretion aldosterone, and aldosterone acts on renal tubule, plays and protects sodium, water conservation, the effect of row's potassium, thereby cause hypervolemia, and blood pressure raises.
Under normal circumstances, because renin secretion is little, blood Angiotensin-Converting is also few, and blood pressure can not regulated to obvious effect.But in the time losing blood greatly, because arterial pressure significantly declines, renal blood flow is reduced, angiotensins generates and increases, and makes blood pressure go up to play an important role to preventing blood pressure excessive descent.The long-term spasm of kidney blood vessel or narrow patient, because renal blood flow reduces, angiotensins generates to increase and can cause renal hypertension.In addition, angiotensins can cause that blood vessel shrinks strongly, regulates but do not participate under normal circumstances blood pressure.When body makes circulating blood volume reduce in the situation such as losing blood, this hormone will significantly raise at blood level, plays a role to keeping circulating blood volume and maintaining arterial pressure.
Radiating immuning analysis technology (RIA) and enzyme-linked immunosorbent assay (ELISA) to the current method of conventional mensuration angiotensinⅠ clinically of angiotensinⅠ at present, but there is many deficiencies in these two kinds of methods, for example RIA exist radioactive contamination, label half life period short, operator is had to radioactive damage, and complex operation, the shortcomings such as time length; And ELISA sensitivity is low, sensing range is narrow; Along with developing rapidly of immuno-labelling technique, various new detection methods emerge in an endless stream, and such as ELISA method, Timed-resolved fluoroimmunoassay, immunofluorescence technique, chemiluminescence are sent out etc.Wherein, chemiluminescence and EIA enzyme immunoassay are combined in this technical growing up by chemiluminescence immune assay (CLIA), is started in the beginning of the eighties, is developed rapidly and applies in the nineties, be current the most responsive skeptophylaxis determination method, there is highly sensitive (detection limit 10
-17-10
-19), can measure the advantages such as material concentration wide ranges, the reagent term of validity are long, simple to operate fast, good stability, safe non-environmental-pollution, at present to be widely applied to basis and clinical medical every field, become the one preferred technique of replacement RIA and ELISA.
Summary of the invention
The problem to be solved in the present invention is to provide chemiluminescence immunoassay immue quantitative detection reagent box of angiotensinⅠ and preparation method thereof, has solved sensitivity low, the defect that sensing range is narrow.
For solving the problems of the technologies described above, the technical solution used in the present invention is: a kind of angiotensinⅠ chemiluminescence immunoassay immue quantitative detection reagent box comprises: Ang I antibody is coated with Ang I, Ang I calibration object, Ang I quality-control product, luminescent solution A liquid and the B liquid of plate, horseradish peroxidase-labeled, 20 times of concentrated washing lotions.
The solid phase carrier of the described coated plate of angiotensinⅠ antibody is the white microwell plate in 96 holes or 48 holes.Described angiotensinⅠ chemiluminescence immunoassay immue quantitative detection reagent box, is characterized in that, described horseradish peroxidase purity requirement RZ >=3.0, activity >=250U/mL.Described luminescent solution A comprises 0.7g/L luminol and 0.165g/L p-iodophenol; Luminescent solution B comprises 0.675g/L urea peroxide.20 times of described concentrated washing lotions comprise 75.5g/L Tris, 120g/L NaCl, 5mL/L Tween-20,1g/LProclin300.
Prepare a method for mentioned reagent box, it is characterized in that comprising the following steps:
1) the coated plate preparation of Ang I antibody
Ang I antibody is diluted to 1-10ug/mL with pH9.6 carbonate buffer solution, in the hole of each microwell plate, add 100uL, in 4 DEG C of refrigerators, place and spend the night, after cleansing solution washing five times, by bovine serum albumen solution, microwell plate is sealed, discard in hole after liquid, ELISA Plate is dry, sealing, obtains through the coated microwell plate of Ang I antibody;
2) horseradish peroxidase-labeled Ang I, obtains Ang I enzyme conjugates;
Utilize sodium periodate oxidation, Ang I and horseradish peroxidase are linked together, form through the Ang of horseradish peroxidase-labeled I;
3) Ang I calibration object;
Ang I antigen is become to series concentration (concentration is respectively 0,0.2,0.5,2,5,12ng/mL) by calibration object diluted, save backup in 2 ~ 8 DEG C.
4) preparation of Ang I quality-control product: in normal human serum, add appropriate Ang I sterling, preparation low value quality-control product (QcL) and high value quality-control product (QcH), the mean value of its concentration is respectively 0.46ng/mL and 10.35ng/mL.
5) preparation luminescent solution A liquid and B liquid;
A liquid is that the 5mmol/LTrisHCl damping fluid of the pH8.6 that contains 0.7g/L luminol and 0.165g/L p-iodophenol is; B liquid is that concentration is the urea peroxide of 0.675g/L, prepares with process water.
6) 20 times of concentrated washing lotions of preparation;
The formula of 20 times of concentrated washing lotions is as follows: 75.5g/L Tris, 120g/L NaCl, 5mL/L Tween-20,1g/LProclin300.
7) assembling: mentioned reagent is assembled into box, is stored in 2～8 DEG C; Comprising each 1 bottle of luminescent solution A, luminescent solution B, 20 times of concentrated washing lotions, Ang I enzyme conjugates, 1 of the coated plate of Ang I, 6 bottles of Ang I calibration objects, 2 bottles of Ang I quality-control products.
8) measured value and the stability of linearity to adopting the kit that makes of the method to carry out physical examination, and to accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
Further described coated plate is 96 holes or the 48 holes white microwell plate that is coated with Ang I antibody.
Kit prepared by the inventive method adopts following concrete form, it comprise white microwell plate, the Ang I antigen of horseradish peroxidase-labeled, the Ang I calibration object of variable concentrations, the luminescent solution A liquid in 96 holes that angiotensinⅠ antibody is coated or 48 holes be luminol and p-iodophenol, luminescent solution B liquid be urea peroxide, 20 times of concentrated washing lotions for formula be 75.5g/L Tris, 120g/L NaCl, 5mL/LTween-20,1g/LProclin300.
ProClin300 is with its broad spectrum of activity, superior compatibility and stability and the hypotoxicity under working concentration thereof, and ProClin300 becomes the desirable efficient antiseptic for diagnostic reagent.Proclin300 antiseptic can be eradicated bacterium, fungi and yeast within the longer time, thereby extends the storage time of product.It is water-soluble guarantees that it can dissolve in required reagent easily.Particularly, ProClin300 anticorrosion on the function of most enzyme or antibody linked reaction without impact, so can interference test indicator.
The angiotensinⅠ immue quantitative detection reagent box of this patent invention, adopts the most responsive at present method of immunity---chemiluminescence immunoassay technology, has a little following: (1) this kit utilizes Chemiluminescence Immunoassay to measure plasma sample.Easy and simple to handle, be applicable to routine clinical detection; (2) highly sensitive, sensitivity for analysis is not higher than 0.05ng/mL; (3) specificity of this product is good, is all less than 1% with the specificity of intersecting of angiotensinⅡ and hypertensin 1-7; (4) precision is good, and withinrun precision is not higher than 15%, and betweenrun precision is not higher than 20%; (5) have good stability, this product can be deposited more than 7 days at 37 DEG C, can deposit 1 year at 2 ~ 8 DEG C; (6) high specificity, reaction fast, can judge testing result in 65 minutes, easy and simple to handle, safe non-environmental-pollution.In addition, the present invention also has the advantages such as the concentration range of sample of detection is wide, reagent term of validity length, good stability.Performance is better than domestic like product, has reached international most advanced level, and the more external product of cost is low.
Brief description of the drawings
Fig. 1 is the measurement result comparison diagram that Bo Aosaisi chemical luminescence reagent kit of the present invention is measured Ang I and radioimmunological kit mensuration Ang I, wherein ordinate is the Ang I value that Bo Aosaisi records, horizontal ordinate is that radioimmunological kit is measured Ang I value, two kinds of method related coefficients (r)=0.9477, straight-line equation y=0.9772x+0.041.
Embodiment
Embodiment 1: prepare angiotensinⅠ chemiluminescence immunoassay immue quantitative detection reagent box
AngiotensinⅠ chemiluminescence immunoassay immue quantitative detection reagent box, it is characterized in that, described kit comprises: Ang I antibody is coated with Ang I, Ang I calibration object, Ang I quality-control product, luminescent solution A liquid and the B liquid of plate, horseradish peroxidase-labeled, 20 times of concentrated washing lotions.
Prepare angiotensinⅠ chemiluminescence immunoassay immue quantitative detection reagent box by following method
(1) coated: Ang I antibody is added in carbonate buffer solution (pH=9.6) and is mixed, in the hole of each microwell plate, add 100uL, in 4 DEG C of refrigerators, place and spend the night, after cleansing solution washing five times, by bovine serum albumen solution, microwell plate is sealed, discard in hole after liquid, ELISA Plate is dry, sealing, obtains through the coated microwell plate of Ang I antibody.
(2) utilize sodium periodate oxidation, Ang I and horseradish peroxidase are linked together, form through the Ang of horseradish peroxidase-labeled I.
The concrete steps of sodium periodate oxidation are as follows:
A is dissolved in HRP in distilled water, adds the 0.06M NaIO4 aqueous solution of isopyknic new preparation, places 30min after mixing in refrigerator, then, with the isopyknic 0.16M ethylene glycol of HRP, under room temperature, places 30min.
B adds the Ang I with HRP equivalent, after mixing, packs bag filter into, to the carbonic acid buffer of 0.05M pH9.5 dialysed overnight at 4 DEG C, then adds the NaBH4 of 5mg/mL, places 2h in refrigerator.
Above-mentioned mixed liquor is added isopyknic saturated ammonium sulfate solution by c, places after 30min centrifugal in refrigerator.
The sediment that d obtains is dissolved in a little 0.02M pH7.4 phosphate buffer, after dialysed overnight, adds isopyknic glycerine to store at-20 DEG C for subsequent use.
3) Ang I calibration object;
Ang I antigen is become to series concentration (concentration is respectively 0,0.2,0.5,2,5,12ng/mL) by calibration object diluted, save backup in 2 ~ 8 DEG C.
4) preparation of Ang I quality-control product: in normal human serum, add appropriate Ang I sterling, preparation low value quality-control product (QcL) and high value quality-control product (QcH), the mean value of its concentration is respectively 0.46ng/mL and 10.35ng/mL.
5) preparation luminescent solution A liquid and B liquid;
A liquid is that the 5mmol/LTrisHCl damping fluid of the pH8.6 that contains 0.7g/L luminol and 0.165g/L p-iodophenol is; B liquid is that concentration is the urea peroxide of 0.675g/L, prepares with process water.
6) 20 times of concentrated washing lotions of preparation;
The compound method of 20 times of concentrated washing lotions is as follows: 75.5g/L Tris, 120g/L NaCl, 5mL/LTween-20,1g/LProclin300.
7) assembling: mentioned reagent is assembled into box, is stored in 2～8 DEG C; Comprising each 1 bottle of luminescent solution A, luminescent solution B, 20 times of concentrated washing lotions, Ang I enzyme conjugates, 1 of the coated plate of Ang I, 6 bottles of Ang I calibration objects, 2 bottles of Ang I quality-control products.
8) measured value and the stability of linearity to adopting the kit that makes of the method to carry out physical examination, and to accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
Illustrate: index detects
Accuracy: kit calibration object and company standard product series are analyzed mensuration simultaneously, with Log (X)-Logit (Y) Model fitting, require two not obvious parallel deviates of dose-response curve (t inspection, | t|<2.447); Taking Ang I company standard product as reference substance, with Log (X)-Logit (Y) Model fitting, the mean value of the measured value of kit calibration object and sign value ratio should be in 0.90 ~ 1.10 scope.
The preparation of company standard product: be serial gradient by highly purified Ang dilution for I (containing the phosphate buffer of bovine serum albumin(BSA)) dilution, through WHO standard product (NIBSC numbering: 86/536) demarcate, its concentration is respectively 0.2,0.5,2,5,12ng/mL, and contrasts as null value using the dilution containing Ang I not.
The linearity of dose-response curve: with Log (X)-Logit (Y) Model fitting, dose-response curve correlation coefficient r absolute value in 0 ~ 12ng/mL concentration range is not less than 0.9900.
Sensitivity for analysis: sensitivity for analysis is not higher than 0.05ng/mL.
Precision: in batch, imprecision (CV%) should be higher than 15.0%; Between batch, imprecision (CV%) should be higher than 20.0%.
Quality-control product measured value: the quality-control product of the high value in replicate determination 10 holes and low value, with Log (X)-Logit (Y) Model fitting, quality-control product measured value should be in allowed band, and the allowed band of QcL and QcH is respectively 0.37～0.55ng/mL and 8.28～12.42ng/mL.
Specificity: cross reaction should meet following table requirement.
Stability: place 7 days for 37 DEG C, measured value should meet every requirement.
Embodiment 2: prepare angiotensinⅠ chemiluminescence immunoassay immue quantitative detection reagent box
Except solid phase carrier is the microwell plate in 48 holes, prepare kit of the present invention with method in the same manner as in Example 1.
Embodiment 3: the using method of kit of the present invention
1. this kit is taken out from 4 DEG C of refrigerators to balance 30 minutes under room temperature (18 ~ 25 DEG C).
2. preparation washing lotion: will concentrate washing lotion with distilled water and dilute (1mL washing lotion adds 19mL distilled water) by 1:20.If concentrated washing lotion has crystallization, can dilute again concentrating after washing lotion is placed in room temperature or 37 DEG C of dissolvings to be crystallized.
3. need to take out appropriate coated slab according to experiment.Blank 1 hole is set, each 2 holes of calibration object, each 10 holes of quality-control product.Every hole adds the each 50 μ L of Ang I calibration object, quality-control product and sample, and blank does not add calibration object, quality-control product and sample.
4. every hole adds Ang I enzyme conjugates 50 μ L, except blank hole.
5. craft or machine vibrate gently and mix for 10 seconds, with cover plate film, plate hole are built, and react 60 minutes at 37 DEG C.
6. throw off cover plate film, sucking-off or pour out after reactant liquor, adds washing lotion to wash five times, and the each every hole of washing lotion amount is no less than 300 μ L, soak time 10 seconds, sucking-off or pour out washing lotion after pat dry.Also can wash with washing plate machine washing.
Every hole add luminescent solution 100 μ L(luminescent solution A and B before use 5min mix by equal-volume), comprise blank hole.
8. room temperature (18 ~ 25 DEG C) is secretly put 5 minutes, measures luminous value on chemical illumination immunity analysis instrument.
The clinical testing of 4 kits of embodiment
The kit of this patent invention has carried out clinical examination, 110 routine clinical samples are chosen in this clinical testing, first with after the test of radioimmunoassay kit, measure with the kit (chemoluminescence method) of this patent invention again, it has been carried out paired t-test, Linear correlative analysis and Chi-square Test and has been evaluated.Result proves, paired t-test statistical study draws P > 0.05; Linear correlative analysis draws r > 0.975, P < 0.001; Chi-square Test statistical study draws sensitivity >95%, specificity >95%.Whether have significant difference, and conspicuousness is relevant, there being good consistance aspect clinical coincidence rate, can promote clinical practice if counting two kinds of product testing results.
In order to determine the reference value of this kit, adopt kit of the present invention to measure to 613 parts of Tianjin human normal plasma's sample, result shows that the normal reference value (term of reference) (2.5% ~ 97.5%) of this kit is:
General food: clinostatism 0.07 ~ 1.50ng/mL
Vertical position 0.30 ~ 5.10ng/mL
Low sodium: clinostatism 0.90 ~ 1.70ng/mL
Vertical position 1.70 ~ 7.40ng/mL
Claims (2)
1. angiotensinⅠ chemiluminescence immunoassay immue quantitative detection reagent box, described kit comprises: Ang I antibody is coated with plate, the Ang I of horseradish peroxidase-labeled, Ang I calibration object, Ang I quality-control product, luminescent solution A liquid and B liquid, 20 times of concentrated washing lotions, it is characterized in that, the coated plate of described angiotensinⅠ antibody is the 48 hole white microwell plates that are coated with Ang I antibody, described horseradish peroxidase purity requirement RZ >=3.0, activity >=250U/mL, described Ang I quality-control product comprises low value quality-control product and high value quality-control product, Ang I is become to series concentration by calibration object diluted, concentration is respectively 0, 0.2, 0.5, 2, 5, 12ng/mL, save backup in 2～8 DEG C, wherein the concentration range of low value quality-control product is 0.46ng/mL, and the concentration range of high value quality-control product is 10.35ng/mL, and described luminescent solution A comprises 0.7g/L luminol and 0.165g/L p-iodophenol, luminescent solution B comprises 0.675g/L urea peroxide, and 20 times of described concentrated washing lotions comprise 75.5g/L Tris, 120g/L NaCl, 5mL/L Tween-20,1g/LProclin300.
2. prepare a method for the kit of described claim 1, it is characterized in that comprising the following steps:
1) the coated plate preparation of Ang I antibody
Ang I antibody is diluted to 1～10 μ g/mL with pH9.6 carbonate buffer solution, in the hole of each microwell plate, add 100 μ L, in 4 DEG C of refrigerators, place and spend the night, after cleansing solution washing five times, by bovine serum albumen solution, microwell plate is sealed, discard in hole after liquid, ELISA Plate is dry, sealing, obtains the 48 hole white microwell plates coated through Ang I antibody;
2) horseradish peroxidase-labeled Ang I, obtains Ang I enzyme conjugates;
Utilize sodium periodate oxidation, Ang I and horseradish peroxidase are linked together, form through the Ang of horseradish peroxidase-labeled I;
3) Ang I calibration object;
Ang I is become to series concentration by calibration object diluted, and concentration is respectively 0,0.2,0.5,2,5,12ng/mL, saves backup in 2～8 DEG C;
4) preparation of Ang I quality-control product: in normal human serum, add appropriate Ang I sterling, preparation low value quality-control product and high value quality-control product, the mean value of its concentration is respectively 0.46ng/mL and 10.35ng/mL;
5) preparation luminescent solution A liquid and B liquid
A liquid is the 5mmol/LTrisHCl damping fluid of the pH8.6 that contains 0.7g/L luminol and 0.165g/L p-iodophenol; B liquid is that concentration is the urea peroxide of 0.675g/L, prepares with process water;
6) 20 times of concentrated washing lotions of preparation
The formula of 20 times of concentrated washing lotions is as follows: 75.5g/L Tris, 120g/L NaCl, 5mL/L Tween-20,1g/LProclin300;
7) assembling
Mentioned reagent is assembled into box, is stored in 2～8 DEG C;
8), to adopting the kit that makes of the method to carry out physical examination, measured value and the stability of the linearity of accuracy, dose-response curve, precision, specificity, sensitivity, quality-control product are measured.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102998465B (en) * | 2012-11-20 | 2015-05-13 | 博奥赛斯（天津）生物科技有限公司 | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for angiotensin (Ang) I, and preparation method of kit |
| CN103604918A (en) * | 2013-11-28 | 2014-02-26 | 中国科学院广州生物医药与健康研究院 | Luminescent substrate, use of luminescent substrate and detection kit containing luminescent substrate |
| CN104634965A (en) * | 2015-02-10 | 2015-05-20 | 深圳市新产业生物医学工程股份有限公司 | Angiotensin I detection reagent kit as well as preparation method and application thereof |
| CN111965369A (en) * | 2020-07-28 | 2020-11-20 | 天津国科医工科技发展有限公司 | Kit and method for detecting angiotensin I in blood plasma |
| CN113985036A (en) * | 2021-11-04 | 2022-01-28 | 南京岚煜生物科技有限公司 | Angiotensin I detection kit and preparation method thereof |
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| US3899298A (en) * | 1972-04-11 | 1975-08-12 | Squibb & Sons Inc | Method and apparatus for angiotensin i determination |
| CN102095866A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆生物科技有限公司 | Chemiluminescence quantitative detection kit for progesterone |
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| JPH01227960A (en) * | 1988-03-09 | 1989-09-12 | Kissei Pharmaceut Co Ltd | Enzyme immunochemical measurement method of angiotensin i by solid phase method |
| JPH01227961A (en) * | 1988-03-09 | 1989-09-12 | Kissei Pharmaceut Co Ltd | Enzyme immunochemical measurement of angiotensin ii |
| JPH022935A (en) * | 1988-06-20 | 1990-01-08 | Tosoh Corp | Angiotensin measuring method |
| JP2002112768A (en) * | 2000-10-04 | 2002-04-16 | Igaku Seibutsugaku Kenkyusho:Kk | Angiostatin specific monoclonal antibody, method for detecting angiostatin using the monoclonal antibody, or the like |
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| US3899298A (en) * | 1972-04-11 | 1975-08-12 | Squibb & Sons Inc | Method and apparatus for angiotensin i determination |
| CN102095866A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆生物科技有限公司 | Chemiluminescence quantitative detection kit for progesterone |
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| Title |
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| 陆以信等.高亲和力抗血管紧张素Ⅱ抗血清的制备――五种免疫原的制备及其免疫效果的比较.《生物化学与生物物理学报》.1977,第09卷(第04期),363-376. * |
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Address after: 300300 building 14, international medical device Industrial Park, No. 16, Wujing Road, economic and Technological Development Zone, Dongli District, Tianjin Patentee after: Tianjin boasaisi Biotechnology Co.,Ltd. Address before: No. 10, Siwei Road, Dongli District, Tianjin 300300 Patentee before: BIOSCIENCE (TIANJIN) DIAGNOSTIC TECHNOLOGY Co.,Ltd. |